PROJECT SUMMARY/ABSTRACT Although mucus is a defensive barrier that is critical for maintaining healthy lungs, excessive mucus is a significant pathological contributor to lung diseases. The disparate faces of mucus function and dysfunction epitomize the need for tightly controlling the predominant macromolecules in airway mucus - the polymeric mucins MUC5AC and MUC5B. In asthma, mucus viscoelasticity increases and causes airflow and mucociliary clearance (MCC) impairments that are not adequately treated. We postulate that by determining specific molecular mechanisms of MUC5AC and MUC5B biosynthesis, we will identify novel mechanisms and potential treatments for improving airway mucus function and dysfunction. We previously demonstrated significant functions for Muc5ac and Muc5b in knockout mice. We now seek to interrogate how Muc5ac and Muc5b function as polymers. Muc5ac and Muc5b assemble by inter-molecular disulfide bonding at their cysteine-rich carboxyl (C-) and amino (N-) termini, resulting in the formation of massive glycopolymers. Recently, we treated allergically inflamed mice with inhaled agents that disrupted mucin polymer disulfide bonds, and we found that mucus viscoelasticity, MCC, and airflow function all improved. While inhaled mucolytics could be useful, there are currently no safe or effective options available for therapeutic use. Thus, there is further need to determine MUC5AC and MUC5B isoform specific polymer functions. We hypothesize that mucus function and dysfunction are dictated by specific mucin isoform expression and disulfide polymerization mechanisms that regulate mucus viscoelastic gel and transport properties. We will test the following hypotheses in thee Specific Aims: 1) that isoform specific mucin polymers mediate AHR and MCC; 2) that polymerization is mediated by disulfide bonds between conserved cysteine residues in the N- and C-termini of MUC5AC/Muc5ac and MUC5B/Muc5b; and 3) that conserved N- and C-terminal cysteines are required for mucin isoform-specific polymerization and function in vivo. We will conduct studies in mucin mutant mice, in human mucus samples, in airway epithelial cell cultures, and with bioengineered Human Small Airway-on-a-Chip devices. Successful completion of the Aims proposed here will further advance our understanding of mucin polymer formation and function in the airways. Our studies could also identify improved strategies for preventing obstruction while preserving defensive mucus functions that are required for lung health.